The present invention is directed to fish containing an oncogenic nucleic acid, to fish tumorgenesis models, to immortal tumor cell lines and to screening for anti-cancer agents.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Animal models of disease states play an important role in identifying the underlying biochemical mechanisms of particular diseases, as well as discovering therapeutic agents to eradicate the disease or otherwise lessen its symptoms. For example, rabbit models of familial hypercholesterolemia, rat models of non-insulin-dependent diabetes mellitus, mouse models of cancer and hamster models for spontaneous atrial thrombosis are known. Additionally, animal models for genetic diseases have arisen spontaneously in a variety of species, including mice, cats and dogs. Working with such large animals poses several drawbacks. For example, many of the animals used in such models are relatively large vertebrates which take up a large amount of research space, are costly to feed and otherwise maintain, have slow reproductive cycles, produce relatively few offspring at one time, and cannot effectively mimic all desired disease states.
Transgenic fish are currently being utilized to develop disease models. A wide variety of fish may be utilized for this purpose. Exemplary fish include teleost fish, such as zebrafish (Danio rerio), medaka (Oryzias latipes), mummichog (Fundulus heteroclitus), killifish (Genus Fundulus), catfish (Genus Ictalurus), such as channel catfish; carp (Genus Cyprinus), such as common carp; and trout or salmon (e.g., Genus Salvelinus, Salmo, and Oncorhiynchus). Zebrafish have become an established model for investigating many facets of development, physiology and disease.
Zebrafish are particularly advantageous because they are small, develop ex utero, and have a short generation time. Zebrafish are economical to maintain in the laboratory environment and are highly fecund; a single female is capable of generating hundreds of offspring per week. At 5 days of age each fish is a free swimming/feeding organism complete with most of the organ systems employed by mammals, such as heart, brain, blood, and pancreas. The zebrafish embryo develops externally and is transparent, allowing direct visualization of cellular and tissue developmental processes as they proceed in vivo, thereby facilitating large-scale genetic and small molecule drug screens. In the past several years numerous publications have reported transgenic fish lines expressing green fluorescent protein (GFP) in cell-type restricted expression patterns (Gong et al., 2001; Kennedy et al., 2001; Long et al., 1997; Moss et al., 1996; Motoike et al., 2000; Park et al., 2000). To date, studies using fluorescent transgenic zebrafish have focused mainly on imaging cells and tissues as they develop. Such transgenic zebrafish lines, in addition to promoting developmental investigations of tissue morphogenesis, facilitate genetic and pharmacological screens by allowing high-resolution imaging of discrete cell populations.
Many of the underlying mechanisms that lead to cancer have yet to be fully understood. Identifying the genes mutated in these diseases will lead to new insights into cancer as a whole. Additionally, using a vertebrate model system in which genetic or chemical suppressors can be identified that inhibit or delay disease progression, or sensitivity to chemotherapy or radiation-induced programmed cell death, will be necessary to identify new drug targets for the development of targeted chemotherapies. For example, a model system is needed, which does not require an a priori knowledge of the specific target. Target elucidation may be accomplished after the modulating target drug or agent is demonstrated safe and effective, which, thus, saves both time and expense in the drug discovery process.
A further understanding of the cellular and molecular genetic features of various disease states such as cancer is needed. An appropriate animal model would be invaluable to extend the understanding of cancer, as well as to enable the development of more effective drugs for treating or preventing cancer. The present invention addresses these needs.